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Identification of Sugars

by Paper Chromatography
Section A4

WHAT IS CHROMATOGRAPHY?
a method of separating and identifying
the components of a complex mixture
differential movement through a twophase system, in which the movement is
affected by a flow of a liquid or a gas
(mobile phase) which percolates through
an adsorbent (stationary phase) or a
second liquid phase

WHAT IS CHROMATOGRAPHY?
Components of a mixture may be interacting
with the stationary phase based on charge,
relative solubility or adsorption
Basic Chromatographic Principles
All chromatographic systems contain:
A stationary phase
A mobile phase
Sample molecules

Type of Chromatography

Applications in the Real World

Why and What is it

Liquid Chromatography

test water samples to look for pollution,

Used to analyze metal ions and


organic compounds in
solutions. It uses liquids which
may incorporate hydrophilic,
insoluble molecules.

Gas Chromatography

detect bombs in airports, identify and


quantify such drugs as alcohol, used in
forensics to compare fibers found on a
victim

Used to analyze volatile gases.


Helium is used to move the
gaseous mixture through a
column of absorbent material.

Thin-Layer
Chromatography

Paper Chromatography

Uses an absorbent material on


detecting pesticide or insecticide residues in
flat glass plates. This is a simple
food, also used in forensics to analyze the
and rapid method to check the
dye composition of fibers
purity of the organic compound.

separating amino acids and anions, RNA


fingerprinting, separating and testing
histamines, antibiotics

The most common type of


chromatography. The paper is
the stationary phase. This uses
capillary action to pull the
solutes up through the paper
and separate the solutes.

PAPER CHROMATOGRAPHY
Paper Partition Chromatography
to emphasize the partitioning of solutes between
a mobile phase and water adhering to the filter
paper during chromatography

PAPER CHROMATOGRAPHY
a method for testing the purity of compounds
and identifying substances
a useful technique because it is relatively quick
and requires small quantities of material

PAPER CHROMATOGRAPHY
Carried out by:
placing samples to be
analyzed on sheets of
filter paper
allowing an organic
solvent to develop
the chromatogram

PAPER CHROMATOGRAPHY
Mobile phase:
ORGANIC SOLVENT

Stationary phase:
THE WATER WHICH IS HELD TO THE CELLULOSE
FIBERS OF THE PAPER BY HYDROGEN BONDING

PROCEDURE
Prepare the developing chamber
Organic solvent: Butanol:Ethanol:Water (52:32:10)

Prepare the Stationary Phase


Spotting the Samples
Developing the Chromatogram
Spray the Chromatogram
Heat the Chromatogram
Identify Spots

PROCEDURE
Prepare the developing chamber
Organic solvent: Butanol:Ethanol:Water (52:32:10)

Prepare the Stationary Phase

Spotting the Samples


Developing the Chromatogram
Spray the Chromatogram
Heat the Chromatogram
Identify Spots

PREPARATION FOR THE STATIONARY PHASE


1. Draw a line, 2cm
away from the edge
of the filter paper
using a pencil.
2. Divided the baseline
into 6 spots.
3. Marked each spot and
labelled according to
the name of the
standard sugars.

PROCEDURE
Prepare the developing chamber
Organic solvent: Butanol:Ethanol:Water (52:32:10)

Prepare the Stationary Phase


Spotting the Samples

Developing the Chromatogram


Spray the Chromatogram
Heat the Chromatogram
Identify Spots

SPOTTING THE SAMPLES


1. Using a capillary
pipette, 4l each of
the given sugar
solutions were
applied.
2. Application is done 6
times for each spot
(drying spot in each
applications).

PROCEDURE
Prepare the developing chamber
Organic solvent: Butanol:Ethanol:Water (52:32:10)

Prepare the Stationary Phase


Spotting the Samples
Developing the Chromatogram
Spray the Chromatogram
Heat the Chromatogram
Identify Spots

DEVELOPING THE CHROMATOGRAM


The paper was
placed in the
chamber
containing the
solvent.
The spots should
not touch the
solvent.

PROCEDURE
Prepare the developing chamber
Organic solvent: Butanol:Ethanol:Water (52:32:10)

Prepare the Stationary Phase


Spotting the Samples
Developing the Chromatogram
Spray the Chromatogram
Aniline acid oxylate spray

Heat the Chromatogram


Identify Spots

PROCEDURE
Prepare the developing chamber
Organic solvent: Butanol:Ethanol:Water (52:32:10)

Prepare the Stationary Phase


Spotting the Samples
Developing the Chromatogram
Spray the Chromatogram

Heat the Chromatogram


Heat in a hot plate until spots become visible.

Identify Spots

PROCEDURE
Prepare the developing chamber
Organic solvent: Butanol:Ethanol:Water (52:32:10)

Prepare the Stationary Phase


Spotting the Samples
Developing the Chromatogram
Spray the Chromatogram
Heat the Chromatogram

Identify Spots

CHROMATOGRAM (1A4-1)

CHROMATOGRAM (1A4-2)

RESULTS
1A4-2

1A4-1
Sugar

Distance traveled
by sugar

Sugar

Distance traveled
by sugar

Galactose

2.6 cm

Galactose

3.6 cm

Glucose

3.3 cm

Glucose

3.9 cm

Fructose

5.1 cm

Fructose

5.5 cm

Maltose

1.0 cm

Maltose

1.4 cm

Sucrose

1.7 cm

Sucrose

2.2 cm

Unknown

1.5 cm

Unknown

2.1cm

Distance traveled by
farthest sugar = 5.1 cm

Distance traveled by
farthest sugar = 5.5 cm

COMPUTING THE Rf VALUE


Rf

distance traveled by unknown


distance traveled by solvent front

i.e. Galactose (1A4-1)


Distance traveled by the sugar = 2.6 cm
Distance traveled by farthest sugar = 5.1 cm
2.6 cm
Rf
5.1 cm

Rf 0.51

1A4-1

RESULTS
1A4-2

Sugar

Rf Value

Sugar

Rf Value

Galactose

0.51

Galactose

0.65

Glucose

0.65

Glucose

0.71

Fructose

1.00

Fructose

1.00

Maltose

0.20

Maltose

0.25

Sucrose

0.33

Sucrose

0.40

Unknown

0.29

Unknown

0.38

DISCUSSION
Mobile phase: Organic solvent system
Butanol: Ethanol: Water (52:32:10) solvent system

Stationary phase: Water molecules embedded in


Whatman filter paper
Color visualization: Reaction of reducing sugars
with aniline acid oxalate and the addition of
heat
Dehydration which produced fulfural from
hydroxymethyl-fulfural

DISCUSSION
Problem: Solvent ran off the paper
distance traveled by unknown
Rf
distance traveled by solvent front

Solution: Change the denominator to the


distance traveled by the farthest sugar
Unknown: Sucrose

SOURCES:
Jack, R. (1995). Basic Biochemical Laboratory
Procedures and Sampling. New York: Oxford
University Press, pages 84-85.
Furfural. Accessed on 07/08/09 at
http://en.wikipedia.org/wiki/Furfural
HMF. Accessed on 07/08/09 at http://
en.wikipedia.org/wiki/Hydroxymethylfurfural